JP2007044041A - Method for screening medicine candidate substance with scrapper protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for screening a medicine. <P>SOLUTION: This method for screening a medicine candidate substance is characterized by adding a tested substance to a screening system containing a ubiquitin-added target protein selected from cell skeleton proteins and synapse-related proteins and Scrapper protein and then measuring the ubiquitin addition degree of the ubiquitin-added target protein with the Scrapper protein, thereby obtaining a compound for changing the ubiquitin addition degree. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for screening a drug candidate substance for diseases such as neurodegenerative diseases and schizophrenia, which utilizes the substrate specificity of the ubiquitin-proteasome pathway.

  Ubiquitin is a highly conserved protein from yeast consisting of 76 amino acids to humans. Ubiquitin is added to a target protein by a cooperative reaction of three enzyme groups, E1 (ubiquitin activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase). The ubiquitinated protein is recognized and degraded by the 26S proteasome, which is a proteolytic enzyme.

  Of these, E3 (ubiquitin ligase) is thought to be most involved in the specificity of the target protein to be ubiquitinated. Recently, a new type of ubiquitin ligase composed of a complex called SCF has been found through research on yeast and the like. This SCF complex type ubiquitin ligase (ubiquitin ligase SCF complex) is a trimer, that is, a subunit protein called Skp1, a subunit protein called Cullin1, a subunit protein called Roc1, And a subunit enzyme called F-box, which is named SCF after the initial letters of each subunit protein. Among these, the Skp1 protein and the Cul1 protein are almost common to any substrate, whereas the F-box protein works differently depending on the substrate, and the substrate specificity of ubiquitin ligase depends on the F-box protein. It is considered to be decided. In the present specification, the intracellular pathway in which the target protein is ubiquitinated and the ubiquitinated target protein is degraded by the proteasome is sometimes referred to as the “ubiquitin-proteasome pathway”. In addition, a system in which a factor related to the ubiquitin-proteasome pathway and a target protein are reacted in a test tube so that the target protein is ubiquitinated and decomposed by the proteasome is sometimes referred to as a “ubiquitin-proteasome system”.

One of the important functions of the ubiquitin-proteasome pathway is removal of abnormal proteins generated in vivo and quantitative regulation by degradation of transcription factors and signal transduction factors. Abnormalities in this pathway are abnormal cell functions. Furthermore, it has been reported to lead to diseases. For example, an association between an abnormality in the ubiquitin-proteasome pathway and carcinogenesis or a neurodegenerative disease has been suggested (Non-Patent Document 1 or 2).
Therefore, if the ubiquitin-proteasome pathway can be controlled in a substrate-specific manner and the degradation of a specific substrate can be suppressed or promoted, it is considered effective for the treatment of diseases. In that case, from the viewpoint of substrate-specific control of the ubiquitin-proteasome pathway, it is conceivable to utilize the substrate selectivity of E3 (ubiquitin ligase), particularly the F-box protein therein. However, the types and substrate specificities of ubiquitin ligases in higher organisms have not yet been fully elucidated, and little is known about the F-box protein involved in the degradation of protein substrates involved in diseases.
“Ubiquitin and neurodegenerative diseases”, “Experimental Medicine”, 2000, Vol. 18, p. 1478-1482 “Molecular Mechanisms of Ubiquitin Disease”, “Proteins / Nucleic Acids / Enzymes”, 1999, Vol. 44, p. 776

  An object of the present invention is to provide a novel method for screening a drug using the substrate specificity of the ubiquitin-proteasome pathway.

  The researchers have made extensive studies to solve the above problems. As a result, we identified the Scrapper protein as an F-box protein of the E3 complex involved in selective degradation of substrates such as KIF and neurofilament, which play an important role in neurons, by the ubiquitin-proteasome pathway. If we can control the degradation of these substrates via the Scrapper protein, we will continue research based on the idea that they will be effective in the treatment of diseases such as schizophrenia and neurodegenerative diseases. The present inventors have succeeded in establishing a new screening system for the drug candidate substances used and have completed the present invention.

That is, the present invention is as follows.
(1) A ubiquitination target protein selected from a cytoskeletal protein and a synapse-related protein, and a test substance are added to a screening system containing a Scrapper protein, and the degree of ubiquitination of the ubiquitination target protein is measured. A method for screening a drug candidate substance.
(2) The screening method according to (1), wherein the screening system is a cell that co-expresses the ubiquitination target protein and the Scrapper protein or an extract of the cell.
(3) The screening method according to (1), wherein the screening system is an in vitro system including the ubiquitination target protein and the Scrapper protein.
(4) The screening method according to (1), wherein the screening system is a non-human mammal expressing a Scrapper protein or a biological sample obtained from the animal.
(5) A ubiquitination target protein selected from cytoskeletal proteins and synapse-related proteins, and a test substance is added to a cell co-expressing a scrapper protein or an extract of the cell, and the ubiquitination target protein is degraded by the proteasome A method for screening a drug candidate substance, characterized by measuring the degree.
(6) A test substance is added to a non-human mammal expressing Scrapper protein or a biological sample obtained from the animal, and a ubiquitination target protein selected from cytoskeletal proteins and synapse-related proteins in the animal or biological sample A method for screening a drug candidate substance, comprising measuring the degree of degradation by a proteasome.
(7) A test substance is added to a screening system containing a ubiquitination target protein selected from cytoskeletal proteins and synapse-related proteins and a Scrapper protein, and the degree of interaction between the Scrapper protein and the ubiquitination target protein is measured. A method for screening a drug candidate substance.
(8) The screening method according to any one of (1) to (7), wherein the ubiquitinated target protein is KIF.
(9) The screening method according to any one of (1) to (7), wherein the ubiquitinated target protein is a neurofilament.
(10) The screening method according to any one of (1) to (7), wherein the ubiquitination target protein is myosin.
(11) The screening method according to any one of (1) to (7), wherein the ubiquitination target protein is RIM.
(12) The screening method according to any one of (1) to (11), wherein the drug candidate substance is a candidate substance for a therapeutic or preventive drug for neurodegenerative disease or schizophrenia.
(13) A screening method for a drug candidate substance, comprising adding a test substance to a nerve cell in which the Scrapper gene is expressed, and measuring the expression level of the Scrapper gene or the release of a neurotransmitter.
(14) A pharmaceutical candidate comprising adding a test substance to a non-human mammal expressing a Scrapper gene or a biological sample obtained from the animal, and measuring the expression level of the Scrapper gene or the release of a neurotransmitter Substance screening method.

  According to the method of the present invention, drug candidate substances useful for the treatment and prevention of diseases such as schizophrenia and neurodegenerative diseases can be efficiently screened.

  The screening method of the present invention comprises adding a test substance to a screening system comprising a ubiquitination target protein selected from cytoskeletal proteins and synapse-related proteins and a Scrapper protein, and in the screening system, ubiquitin of the ubiquitination target protein This is a method for screening a drug candidate substance, characterized by measuring the degree of oxidization, the degree of degradation of the ubiquitinated target protein by the proteasome, or the degree of interaction between the ubiquitinated target protein and the Scrapper protein.

Here, the Scrapper protein is an F-box protein contained in the SCF complex of ubiquitin ligase E3, which means a protein that binds to the above ubiquitinated target proteins and ubiquitinates them, and is also called FB1 protein. . Specific examples of the Scrapper protein include a protein having the amino acid sequence of SEQ ID NO: 2 (human) or 4 (mouse). The Scrapper protein that can be used in the present invention may be a protein derived from another organism as long as it binds to the ubiquitination target protein and can ubiquitinate them, and in the amino acid sequence, It may be a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added. Here, the term “several” is preferably 2 to 50, more preferably 2 to 20, further preferably 2 to 10, and particularly preferably 2 to 5.
The scrapper protein does not necessarily need to be a full-length protein, and a partial fragment thereof may be used as long as it has the above properties.
Moreover, in order to confirm the amount of the Scrapper protein, for example, a tag protein may be added. Examples of the tag protein include a FLAG tag, Myc tag, GFP tag, EGFP tag, and YFP tag.

The target protein of the Scrapper protein used in the screening method of the present invention is selected from cytoskeletal proteins and synapse-related proteins.
Specifically, cytoskeletal proteins that are target proteins of the Scrapper protein are classified into motor proteins and intermediate filaments. The motor protein means a series of proteins involved in vesicular transport and the like, and examples thereof include myosin, kinesin, and dynein. Among these, kinesin or myosin is preferable. Kinesins include many proteins as the kinesin superfamily (KIF), among which KIF1A, KIF1B, and KIF5 are particularly preferable. Here, alphabets in protein names and gene names mean isoforms. For example, the human KIF1 gene transcribes and expresses KIF1A, Ba, and Bb isoforms. For those that do not describe the alphabet, if isoforms are present, they may mean their collection.
Hereinafter, for KIF1A, 1B (a, b), 2, and 5A, the base sequence of the gene and the amino acid sequence of the protein are indicated by the Accession number of GenBank. However, these KIFs are not limited to proteins with the following amino acid sequences, as long as they are ubiquitinated by the Scrapper protein, or those derived from other organisms, or in these sequences, one or several amino acids are substituted, It may be a protein having an amino acid sequence that is deleted, inserted, or added. Partial fragments may also be used. Moreover, what added the said tag protein to these proteins or partial fragments may be used.

Base sequence Amino acid sequence Human KIF1A NM # 004321 NP # 004312
Human KIF1Ba NM # 183416 NP # 904325
Human KIF1Bb NM # 015074 NP # 055889
Mouse KIF1A NM # 008440 NP # 032466
Mouse KIF1B NM # 207682 NP # 997565
Mouse KIF2 NM # 008442 NP # 032468
Human KIF5A NM # 004984 NP # 004975
Mouse KIF5A NM # 008447 NP # 032473
For human KIF5A, SEQ ID NO: 5 (base sequence), 6 (amino acid sequence), for mouse KIF1B, SEQ ID NO: 14 (base sequence), 15 (amino acid sequence), for mouse KIF5A, SEQ ID NO: 16 (Base sequence) and 17 (amino acid sequence) are also described in the sequence listing.
Among myosins, myosin IIB and myosin Va are particularly preferred.
Hereinafter, for myosin IIB, IIA and myosin Va, the base sequence of the gene and the amino acid sequence of the protein are indicated by the Accession number of GenBank. However, these myosins are not limited to proteins with the following amino acid sequences, as long as they are ubiquitinated by the Scrapper protein, or those derived from other organisms, or in these sequences, one or several amino acids are substituted, It may be a protein having an amino acid sequence that is deleted, inserted, or added. Partial fragments may also be used. Moreover, what added the said tag protein to these proteins or partial fragments may be used.
Base sequence Amino acid sequence
Human Myh10 (Myosin IIB) NM # 005964 NP # 005955
Human Myh9 (Myosin IIA) NM # 002473 NP # 002464
Mouse Myosin Va NM # 010864 NP # 034994
Mouse myosin Va is also described in the sequence listing as SEQ ID NOs: 9 (base sequence) and 10 (amino acid sequence).

Examples of the intermediate filament include neurofilament (NF), keratin, desmin, glial fibrillary acidic protein (GFAP), lamin, and nestin, among which neurofilament is preferable.
The neurofilament is composed of a trimer of neurofilaments H, M, and L. Hereinafter, with respect to the neurofilaments H, M, and L, gene base sequences and protein amino acid sequences are indicated by GenBank Accession numbers. However, these neurofilaments are not limited to proteins with the following amino acid sequences, and as long as they are ubiquitinated by the Scrapper protein, one or several amino acids are substituted in those derived from other organisms or in these sequences. It may be a protein having an amino acid sequence deleted, inserted, or added. Partial fragments may also be used. Moreover, what added the said tag protein to these proteins or partial fragments may be used.
Base sequence Amino acid sequence Human NF-H NM # 021076 NP # 066554
Human NF-M NM # 005382 NP # 005373
Human NF-L NM # 006158 NP # 006149
In addition, about human NF-M, it described also in the sequence table as sequence number 7 (base sequence) and 8 (amino acid sequence).

KIF1B is highly expressed in the nervous system and is a motor protein and a synapse-related protein. In addition, for example, synaptic vesicle proteins RIM1, synaptotagmin, Munc-13, and synaptic membrane proteins such as SNAP-25 may be the target molecules of Scrapper (RS Zucker, WG Regehr, Annu Rev
Physiol 64, 355-405 (2002) and TC Sudhof, Annu Rev Neurosci 27, 509-47 (200
Four)).
RIM1 is an abundant protein in the active zone and was discovered in 1997 by Dr. Sudhof (USA) (Y. Wang, M. Okamoto, F. Schmitz, K. Hofmann, TC).
Sudhof, Nature 388, 593-8 (Aug 7, 1997)). It is the most analyzed protein among the active zone proteins currently found. Studies on mice that disrupt the RIM1 gene reveal that RIM1 plays an important role in learning, memory formation, and the development of emotions such as fear.
Hereinafter, for RIM1, the base sequence of the gene and the amino acid sequence of the protein are indicated by GenBank Accession numbers. However, these RIM1 is not limited to the protein of the following amino acid sequence, as long as it is ubiquitinated by the Scrapper protein, or those derived from other organisms, or in these sequences, one or several amino acids are substituted, It may be a protein having an amino acid sequence that is deleted, inserted, or added. Partial fragments may also be used. Moreover, what added the said tag protein to these proteins or partial fragments may be used.

Base sequence Amino acid sequence Human RIM1 NM # 014989 NP # 055804
Mouse RIM1 NM # 001012625 NP # 898839
In addition, about human RIM1, it described also in the sequence table as sequence number 18 (base sequence) and 19 (amino acid sequence).

Motor proteins such as KIF are known to be involved in the transport of synaptic vesicles (Curr Opin Neurobiol. 2004 Oct; 14 (5): 564-73). Therefore, it is considered that by suppressing the degradation, the transport activity of synaptic vesicles is enhanced, and the release of glutamic acid contained in the synaptic vesicles is promoted.
Actually, as shown in the Examples described later, the expression of KIF is increased in the Scrapper gene knockout mouse, and the amount of glutamate released from the synaptic vesicle is also increased. From these facts, it is considered that a drug effective for the treatment and prevention of schizophrenia can be obtained by screening using a scraper protein and a motor protein such as KIF.
KIF1b is a causative gene of Charcot-Marie-Tooth (CMT), a neurodegenerative disease of the spinal cord, and it has been reported that KIF1b is decreased due to gene mutation in the family of CMT (Cell, 105, 587 -597, 2001). Therefore, it is considered that a therapeutic agent for CMT can be obtained by suppressing the degradation of KIF1b via Scrapper.

On the other hand, intermediate filaments such as neurofilaments can be used as targets for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). That is, it is known that degenerated neurofilament accumulates in the brainstem in ALS patients (Annu Rev Neurosci. 2004; 27: 723-49). Therefore, it is considered that neurodegenerative diseases such as ALS can be treated by degrading the degenerated neurofilament by promoting ubiquitination via the Scrapper protein.
In addition to the CMT and ALS listed above, neurodegenerative diseases include polyglutamine diseases, which are hereditary diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's chorea. These show specific symptoms (dementia, ataxia, tremor, weakness, etc.). Further, it has been found that inclusion bodies, which are protein aggregates having an abnormal structure, accumulate in nerve cells as a common pathology of neurodegenerative diseases.
Proteins synthesized in cells become various enzymes and components of the body, but only those with the correct three-dimensional structure can perform individual functions. Even if it is made according to the blueprint, about one third of the protein is made without having the correct structure and is immediately destroyed, and the role of distinguishing and destroying this abnormal protein is a group of cells in the ubiquitin-proteasome pathway The inner protein is responsible.
Recently, many inclusion bodies found in neurodegenerative diseases have been found to contain proteins of the ubiquitin-proteasome pathway. This suggests that one of the causes of neurodegenerative diseases is that the ability to identify and destroy abnormal proteins is attenuated. By controlling the function of Scrapper, a protein of the ubiquitin-proteasome pathway, it is thought that abnormal proteins are properly managed and inclusion bodies are not formed, leading to prevention and treatment of neurodegenerative diseases.

Specifically, as a screening method of the present invention, in vitro ubiquitination measurement system, intracellular ubiquitination measurement system, intracellular degradation measurement system, measurement system using animals, in vitro interaction system, etc. The method of evaluating a compound is mentioned. However, the measurement system is not limited as long as the Scrapper protein and the ubiquitinated target protein are used.
Hereinafter, the measurement system will be specifically described.

(I) In vitro ubiquitination measurement system (I) -1 Add ubiquitin ligase (Scrapper-Skp1-Cullin 1-Roc1 complex) and target protein (KIF, etc.) in the presence of ubiquitin and add ATP, E1, and E2. Incubate (eg, 37 ° C., 30 minutes) and observe the ubiquitination of the target molecule. Among them, the degree of ubiquitination is compared and examined when the test substance is added and when it is not added. Examples of the method for examining the degree of ubiquitination include a method of performing electrophoresis and observing whether the target molecule has undergone a change in molecular weight due to ubiquitination. Further, when the amount of protein is small, a change in molecular weight can be detected with an antibody against the target molecule or ubiquitin (Western analysis).
Scrapper, Skp1, Cullin 1, Roc1, target protein, E1, and E2 added to this system can be proteins prepared by genetic recombination. Examples of genetic recombination include, for example, a DNA having the full-length or partial base sequence of the following gene that is incorporated into a vector that can be expressed in a host cell such as Escherichia coli or a mammalian cell, introduced into the host cell, and expressed. Can be obtained by purification. Ubiquitin may be a commercially available product or may be prepared by gene recombination. Hereinafter, for the protein added to this system, the base sequence of the gene and the amino acid sequence of the protein are shown by SEQ ID NO: or GenBank Accession No.
Base sequence Amino acid sequence Human Scrapper SEQ ID NO: 1 SEQ ID NO: 2
Mouse Scrapper SEQ ID NO: 3 SEQ ID NO: 4
Human Skp1 NM # 170679 NP # 733779
Human Cullin 1 NM # 003592 NP # 003583
Human Roc1 NM # 005406 NP # 005397
Human E1 NM # 003334 NP # 003325
Human E2 U39318 AAA91461
Ubiquitin NM # 021009 NP # 066289
NEDD8 NM # 006156 NP # 006147
APPBP1 NM # 003905 NP # 003896
Uba3 NM # 003968 NP # 003959
UbcH12 NM # 003969 NP # 003960

(I) -2 When the target molecule is ubiquitinated by the above method, it is preferable to examine whether the target molecule is further reduced (decomposed) by adding a proteasome. Examples of the examination method include a method of performing electrophoresis and observing a change in the amount of the target protein. At that time, if the amount of protein is small, a change in the amount of the target protein can be detected with an antibody against the target molecule (Western analysis). Alternatively, it is possible to detect using RI or a fluorescently labeled target protein.
As the proteasome, for example, a purified proteasome (# BB-E-350, manufactured by MBL) can be used.

(II) In vivo degradation measurement system (II) -1 Add a test substance directly to cultured cells that co-express crapin protein and ubiquitinated target proteins such as KIF, or add a test substance to the cell lysate Compare the change in the amount of target protein with that without adding the test substance. In this case, since it is considered that the cell contains all components including the proteasome, the influence of the test substance on the degradation of the target protein can be evaluated. Analysis can be performed in the same manner as (I) -2.
Here, the cell to be used may be any cell that expresses the Scrapper gene, but a cell that overexpresses the Scrapper gene is preferred. When the Scrapper gene is overexpressed, for example, the Scrapper gene can be incorporated into a vector that can be expressed in a mammalian cell, and the gene can be introduced into the mammalian cell.

(II) -2 In the method of (II) -1, a proteasome inhibitor (eg, MG132) can be further added to detect the degree of ubiquitination when the proteasome is blocked. In addition to the same method as described above, ubiquitin antibodies (Western analysis) and labeled ubiquitin (ELISA) can be used as analysis methods.

(III) Measurement system using animals
Using a non-human mammal that expresses the Scrapper gene or a biological sample obtained from the animal, extract the tissue (such as nerve tissue) that you want to compare between when the test substance is given and when the test substance is not given To examine changes in the amount of target protein. It is preferable to use a transgenic non-human mammal overexpressing the Scrapper gene or a biological sample obtained from the animal. Analysis can be performed in the same manner as (I) -2. Examples of biological samples include various organs, tissues, cells, extracts thereof, tissue specimens, cultured cells, body fluids such as blood and urine, and the like. Since the non-human animal of the present invention can be suitably used as a model of, for example, a cranial nervous system disease, a brain slice specimen, a cerebral nerve cell, etc. are particularly preferably used for the analysis of such a disease.
A transgenic animal that overexpresses the Scrapper gene can be produced by the method described in the Examples.

(IV) Interaction Measurement System The screening method of the present invention may be a method of measuring the interaction between the Scrapper protein and the target protein in vitro and selecting a compound that inhibits or activates the interaction between the two. Examples of the in vitro interaction measurement system include a pull-down assay using a scrapper protein and a target protein, and a detection method using a surface plasmon resonance phenomenon. When performing a pull-down assay, the Scrapper protein and the target protein are incubated in vitro, and the complex is recovered with an antibody against one of the proteins, or when this protein is a fusion protein, an antibody against the peptide tag to be fused, an affinity column, etc. Thereafter, the interaction between both proteins can be evaluated by detecting the other protein that binds to the protein. Screening can be performed by adding a test substance to this system and selecting a substance that affects the interaction. In this system, Scrapper protein and substrate protein expressed and purified in E. coli and mammalian cells can be used. Either of them may be labeled with a radioactive substance or the like. An in vitro system using the surface plasmon resonance phenomenon can be performed, for example, by using a biosensor such as BIAcore (registered trademark). Furthermore, as another screening system, a system that detects by fluorescence can also be used (Fluorescence Resonance Energy Transfer (FRET)).

It is also possible to measure the interaction between the Scrapper protein and the target protein in a cell system. For example, the interaction in the cell system can be evaluated by immunoprecipitation or the 2-hybrid method. That is, after culturing cells expressing the Scrapper protein and the target protein, and recovering the cells, the complex is recovered with an antibody against one of the proteins, and then the other protein is detected using an antibody against the protein. Thus, the interaction between the two proteins can be detected, and the influence of the test substance on the interaction can be evaluated. The 2-hybrid method is, for example, “MATCHMARKER Two-Hybrid System”, “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER One-Hybrid”.
"System" (both manufactured by Clontech), "HybriZAP Two-Hybrid Vector System" (manufactured by Stratagene) and the like can be used.
In addition, when Scrapper is present, the ubiquitinated target protein is degraded by the proteasome, and the interaction between the ubiquitinated target protein and Scrapper cannot be detected. However, as shown in Examples 5 and 14 described later, The presence of a compound that inhibits degradation makes it possible to detect the interaction between the two. Therefore, a compound that promotes the interaction (complex formation) of both in this system can also be screened.

(V) A system using the expression level of the Scrapper gene and the release amount of the neurotransmitter as an index As shown in the examples below, a knockout mouse prepared by disrupting the Scrapper gene (hereinafter referred to as “Scrapper gene knockout mouse”) Or, it is sometimes referred to as “Scrapper knockout mouse.” In addition, it was found that the release of glutamate is enhanced in the nerve cells of the knockout (sometimes referred to as “KO”). On the contrary, as shown in the Examples described later, it was found that the release of glutamic acid was reduced in neurons overexpressing the Scrapper gene. This suggests that the Scrapper gene is involved in the neurotransmitter release mechanism. Neurotransmitters include acetylcholine, amino acids, amines, peptides, etc., but amino acids are preferred. Amino acids include glutamic acid, glycine, GABA and the like, with glutamic acid being preferred. Therefore, it is considered that a compound that changes the expression level of the Scrapper gene can be a candidate substance for a therapeutic drug such as schizophrenia. Therefore, the screening method of the present invention includes a method of adding a compound to cells expressing the Scrapper gene and selecting a compound that changes the expression level of the Scrapper gene, or a gene construct in which a reporter gene is linked to the Scrapper gene promoter. A method of selecting a compound that changes the expression of the reporter gene by adding the compound to the introduced cell, a neuron that expresses the Scrapper gene, a non-human mammal, or a biological sample obtained from the animal, and the neurotransmission For example, a method of selecting a compound by quantitatively measuring the amount of a substance may be used.

One or a plurality of screening systems as described above can be used to obtain drug candidate compounds such as therapeutic agents for central diseases such as therapeutic agents for schizophrenia and therapeutic agents for neurodegenerative diseases.
The type of test substance used for screening is not particularly limited, and examples thereof include peptides, proteins, non-peptide compounds, synthetic compounds, polymers, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. These substances may be novel substances or known substances. Moreover, you may add in the state of the compound library containing several types of compounds. The addition amount of the test substance can be appropriately selected according to the administration subject, administration method, properties of the test substance, and the like.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to the following. In the examples, the mouse scraper was used as the scraper.
It describes about the antibody with respect to Scrapper protein used in the Example, and another antibody. An antibody against Scrapper protein was obtained by the following procedure. Rabbit polyclonal antibodies were prepared by expressing Scrapper amino acids 321 to 380 in E. coli as GST fusion proteins and immunizing rabbits using the purified antigens as antigens. Further, an affinity purified product of the antibody was used in the experiment. Hereinafter, the commercially available antibody used and the dilution ratio when the antibody is used for Western blotting are described.
Anti-KIF1A antibody (mouse monoclonal antibody: manufactured by BD) 1: 1000
Anti-Kinesin heavy chain (KIF5) antibody (mouse monoclonal antibody: manufactured by Chemicon) 1: 1000
Anti-Glutamate antibody (rabbit polyclonal antibody: Sigma) 1: 2000
Anti-Phosphatysil CREB antibody (rabbit polyclonal antibody: Cell Signaling) 1: 1000
Anti-CREB antibody (rabbit polyclonal antibody: Cell Signaling) 1: 1000
Anti-Actin antibody (rabbit polyclonal antibody: Sigma) 1: 200
Anti-MyosinIIA antibody (rabbit polyclonal antibody: Sigma) 1: 1000
Anti-MyosinIIB antibody (rabbit polyclonal antibody: Sigma) 1: 200
Anti-MyosinVa antibody (rabbit polyclonal antibody: Sigma) 1: 1000
Anti-PSD-95 antibody (mouse monoclonal antibody: manufactured by UBI) 1: 50000
Anti-Skp1 antibody (mouse monoclonal antibody: manufactured by Zymed) 1: 1000
Anti-Cullin1 antibody (mouse monoclonal antibody: manufactured by UBI) 1: 2000
Anti-FLAG antibody (mouse monoclonal antibody: Sigma) 1: 1000
Anti-GFP antibody (mouse monoclonal antibody: manufactured by BD, manufactured by MBL) 1: 1000
Anti-Ubiquitin antibody (mouse monoclonal antibody: MBL) 1: 200
Anti-KIF2 antibody (mouse monoclonal antibody: manufactured by BD) 1: 250
Anti-KIF1B antibody (goat polyclonal antibody: Santa Cruz) 1: 1000
Anti-MAP2 antibody (mouse monoclonal antibody: Sigma) 1: 1000
Anti-Dynein antibody (mouse monoclonal antibody: manufactured by Chemicon) 1: 1000
Anti-Neurofilament-H antibody (mouse monoclonal antibody: Sigma) 1: 1000
Anti-Neurofilament-M antibody (mouse monoclonal antibody: Sigma) 1: 1000
Anti-Neurofilament-L antibody (mouse monoclonal antibody: Sigma) 1: 1000
Anti-RIM1 antibody (mouse monoclonal antibody: manufactured by BD) 1: 1000

Example 1: Generation of Scrapper gene knockout (KO) mice
General techniques relating to DNA manipulation and the like were performed according to known techniques described in Molecular Cloning, A Laboratory Manual, 3rd edition (Sambrook and Russell, Cold Spring Harbor Laboratory (2001)), unless otherwise specified.
(1) Screening of Scrapper genomic DNA from genomic libraries
In order to obtain genomic DNA for creating a targeting vector for the production of KO mice for the Scrapper gene, a ³² P-labeled probe was made using Scrapper cDNA (SEQ ID NO: 3), and the 129 / Sv FixII-phage genomic library (Stratagene) Screening). Specifically, it was prepared using [α- ³² P] dCTP (Amersham, Redivue, 50 μCi, 3000 Ci / mmole) and a labeling kit (Promega, Prime-α-Gene Labeling System) and spin column (Amershan Pharmacia Biothech Unreacted reagents were removed using MicroSpin Colum SP300HR. RI uptake was measured and evaluated by the Cherenkov method.
Composition of reaction solution according to protocol
Labeling 5x Buffer 10ml
unlabeled dNTP (A, G, T) 2ml 20mM each
template 25ng 500ng / ml
Nuclease Free BSA 2ml 400mg / ml
[α- ³² P] dCTP, 50μCi, 3000Ci / mmole 5ml 333nM
Klenow Fragment 5units 100U / ml
Total volume 50ml
Stop at 1 minute at 95 ° C. for 2 minutes at room temperature.
Of the fragments obtained by NsiI / EcoRI digestion from the subcloned Scrapper cDNA into pCR2.1-TOPO vector (K4500-01, manufactured by Invitrogen) as a template, a fragment of about 300 bp (5'-side) 1 to the middle of the 5th exon) was used for screening to obtain Scrapper genomic DNA.

(2) Mapping of the Scrapper gene Restriction enzyme mapping and sequencing were performed on the Scrapper genomic clone, clone 1, which contains the second exon, and clone 3, which contains the fourth exon to the seventh exon, and the Scrapper genome sequence was analyzed.

(3) Construction of targeting vector The targeting vector was designed to lack the third exon of the Scrapper gene, and as a result, to lack the F-box region of the Scrapper gene.
Using a 7.4 kb SpeI-NotI fragment as the 5 'adjacent homologous region and a 5.8 kb SalI-NdeI fragment as the 3' adjacent homologous region, a 3.2 kp region containing the third exon sandwiched between the two adjacent homologous regions Was replaced with a neo cassette (neomycin resistance gene), and a targeting vector was constructed. In addition, DT-A (diphtheria toxin-A) gene was added to the 3 ′ end of the 3 ′ adjacent homologous region for negative selection in later screening. The construction of the targeting vector is shown in FIG.

(4) Introduction of targeting vector into ES cells The targeting vector obtained in (3) was introduced into ES cells, and target ES clones were selected based on G418 drug resistance. These procedures were performed as used in reproductive engineering.

(5) Southern analysis and PCR screening From ES clones that were positive in (4), clones in which homologous recombination that suits the purpose occurred were selected by Southern blotting and PCR. Confirmation of homologous recombination was carried out by Southern blotting using KpnI / HindIII digestion for Left arm, KpnI-SpeI as a probe, and NcoI digestion for Right arm, and NdeI-NcoI as a probe. For those in which recombination occurred, there were signal changes of 10.5 kb → 9.4 kb and 9.7 kb → 8.6 kb, respectively.
The following three primers were used for confirmation by PCR.
SCR-KO-3 ';5'-CCTCAATGTCCCTCTGGAAATC-3' (SEQ ID NO: 11)
SCR-KO-5 ';5'- CGGAGACTGAGGACATTTTTGG-3 (SEQ ID NO: 12)
SCR-KO-Neo; 5'-TGCATCTGCGTGTTCGAATTCG-3 '(SEQ ID NO: 13)

(6) Blast cyst injection and production of chimeric mouse Heterozygous ES cells were injected into blast cysts of C57BL / 6J strain mice to prepare chimeric mice.

(7) Breeding and mating of chimeric mice Males of chimeric mice obtained in (6) were mated with females of C57BL / 6J strain mice to obtain offspring.

(8) Confirmation of germline transmission (Southern, PCR)
As in (5), the germline transmission was confirmed by Southern blotting and PCR.

(9) Obtaining homozygous mice by crossing heterozygous mice Crossing male and female individuals determined to be heterozygous in (8), and determining the genotype by PCR as in (5) to obtain homozygous mice (Figure 1, lower left). Further, the disappearance of the Scrapper protein was confirmed by Western blotting using an antibody (lower right diagram in FIG. 1). By the above operation, mice lacking the Scrapper gene were obtained.

Example 2: Production of Scrapper gene transgenic mice
A Scrapper cDNA (SEQ ID NO: 3) encoding the 1-438th amino acid of Scrapper protein was subcloned into pEGFPC2 vector (BD Clontech), and EGFP-Scrapper added with AscI restriction enzyme sequence and FseI restriction enzyme sequence before and after The fragment was amplified by PCR. Separately, the MM403 (Mayford et al., Proc. Natl. Acad. Sci. USA. 1996 Nov 12; 93 (23): 13250-5) vector was modified and the AscI-FseI restriction enzyme sequence was inserted into the NotI site. The EGFP-Scrapper fragment was ligated to a vector in which MM403 was modified using the AscI-FseI restriction enzyme sequence. The obtained vector was cleaved with SfiI and subjected to electrophoresis using a low melting point agarose gel to cut out an agarose gel containing an approximately 11 kb transgene fragment. The excised agarose was digested with Gelase (G09050, manufactured by EPICENTRE), and the CamKII promoter-EGFP tag-Scrapper-SV40 polyA transgene was separated and purified. A purified transgene is introduced into a C57BL6J mouse embryo, and a transgenic mouse overexpressing the Scrapper gene (hereinafter sometimes referred to as “Scrapper gene transgenic mouse” or “Scrapper transgenic mouse”. May be referred to as “Tg”). FIG. 2 shows the results of Southern blotting and Western blotting.

Example 3: Identification of target molecules KIF5, KIF1A, KIF1B (two-dimensional electrophoresis and Western blotting of KO and Tg mouse proteins)
Using the Scrapper KO mouse and Tg mouse obtained in Example 1 and Example 2, the target molecule of Scrapper was identified by analyzing quantitative changes in protein by two-dimensional electrophoresis and Western blot. After the mouse was dislocated from the cervical spine, the brain was removed and weighed. Extracted brain with Polytron homogenizer together with an extraction buffer (7M urea, 2M thiourea, 2% CHAPS, 40 mM DTT, 2% IPG buffer pH3-10, Complete EDTA (-) (Roche)) 20 times the brain weight After 3,000 revolutions and 10 strokes, the mixture was centrifuged at 15,000 rpm for 20 minutes at 4 ° C. in a cooling small centrifuge. The obtained supernatant was collected, and protein quantification was performed by the Bradford method (Bio-Rad Protein Assay Dye Regent Concentrate, manufactured by Bio-Rad). Based on the quantified protein concentration, an SDS protein sample was prepared so as to be 0.5 μg / μl or 0.05 μg / μl. SDS was performed by the method of Laemmli et al. After applying 10 μl of each sample to each lane using 8% polyacrylamide gel by the method of Laemmli et al., SDS-PAGE was performed, and the protein in the gel was further transferred to a PVDF membrane (Millipore). Transcription was performed according to Trnovsky et al. Biotechniques 13 (5), 800-804 (1992). Next, the PVDF membrane was subjected to Western blotting. After blocking for 1 hour in TBST (50 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween20) in which 5% skim milk (Wako) was dissolved, the anti-KIF1A antibody (manufactured by BD transduction) was used as the primary antibody. ), Anti-KIF1B antibody (manufactured by Santa Cruz) and anti-KIF5A antibody (manufactured by Chemicon) were each incubated at 4 ° C. overnight at a concentration of 1 μg / ml for reaction. After the reaction, the membrane was washed with TBST, and reacted for 30 minutes at room temperature in a solution diluted with a peroxidase-conjugated anti-mouse or anti-goat antibody (Jackson) 5000 times. After the membrane was again washed with TBST, a peroxidase luminescent substrate (Western blotting detection regents: manufactured by Amersham) was added to perform a chemiluminescent reaction, and the film (Hyperfilm, manufactured by Amersham) was exposed to light and detected. By this operation, the amounts of KIF1A, KIF1B, and KIF5 proteins in Scrapper Tg mice and KO mice were detected, and the amounts were compared with those in normal mice. The results are shown in FIG. The result of Western blotting is shown in the upper part of FIG. 3, and the band density is shown in the lower part of FIG. It was detected that KIF molecules increased in KO mice than in normal mice and decreased in Tg mice. In KO mice, KIF molecules are increased due to scrapper deficiency compared to normal mice, and in Tg mice, KIF molecules are thought to decrease due to more degradation of scrapper overexpression. That is, it was shown that Scrapper has a function of decomposing KIF molecules, and KIF1A, KIF1B, and KIF5 are Scrapper target molecules.

Example 4: Analysis of interaction between Scrapper and KIF5, KIF1A, KIF1B, RIM1 (immunoprecipitation from mouse brain)
In order to examine the interaction between Scrapper and KIF or RIM1, immunoprecipitation was performed on the brain extract of a mouse (C57BL / 6J or ICR) using the aforementioned Scrapper antibody. Three mice were dislocated after cervical dislocation, the brain was removed, homogenized with 10 ml of bufferA (50 mM Tris pH 8.0, 100 mM NaCl, 1% TritonX-100, 10 μg / ml leupeptin, 10 μg / ml PMSF), at 4 ° C., Centrifugation was performed at 100,000 × g for 30 minutes to obtain a mouse brain extract. The solution was divided into two equal parts, each of which was preincubated with 10 μl of protein A sepharose (Amersham) previously incubated with 10 μl of anti-Scrapper antibody, and serum obtained from rabbits before immunization with Scrapper as a negative control. The mixture was mixed with 10 μl of protein A sepharose (Amersham) and incubated at 4 ° C. overnight. After incubation, protein A sepharose was washed 4 times with 1 ml of buffer A, 50 μl of buffer A and 50 μl of 2 × Laemmli buffer were added, and the mixture was heated at 98 ° C. for 3 minutes for SDS conversion. SDS-PAGE was performed in the same manner as in Example 3. However, the sample volume was 10 μl per lane. Western blotting was performed in the same manner as in Example 3 to detect the binding of Scrapper and KIF5, KIF1A, KIF1B, and RIM1 in the mouse brain. As shown in FIG. 4, KIF5, KIF1A, KIF1B, and RIM1 co-precipitated with Scrapper, indicating that KIF5, KIF1A, KIF1B, and RIM1 bind to Scrapper. In contrast, when Western blotting was performed using an anti-KIF2 antibody (BD) and an anti-Dynein-M antibody (Chemicon) as primary antibodies, KIF2 and Dynein-M coprecipitated with Scrapper. It is clear that the binding of KIF1A, KIF1B, RIM1 and Scrapper is specific.

Example 5: Analysis of interaction between Scrapper and KIF1B (immunoprecipitation from 293 cells)
Scrapper cDNA added with FLAG tag (SEQ ID NO: 3) and KIF1B (mouse) cDNA added with GFP tag (SEQ ID NO: 14) were respectively pCMV-Tag4 (Stratagene) and pBOSS (Nucleic Acids)
Research, Vol. 18, No. 17, 5322, 1990) and used as a mammalian cell expression vector. Scrapper and KIF1B, or KIF1B only as a negative control, was expressed in 293 cells using Lipofectamine2000 (Invitrogen). After 1 day, MG132 (manufactured by Peptide Institute) was used to inhibit degradation in the ubiquitin-proteasome system. Was allowed to act at a concentration of 10 μM for 24 hours, and then cells were collected and used in the following experiments. 300μl of bufferA (50mM Tris pH8.0, 100mM NaCl, 1% TritonX-100, 10μg / ml leupeptin, 10μg / ml PMSF) is added per 6cm dish of cells, and ultrasonically disrupted at 4 ℃ Centrifugation was performed at 100,000 xg for 15 minutes to obtain a cell extract. The solution was mixed with 1 μg of anti-GFP antibody and 10 μl of protein A Sepharose (manufactured by Amersham) previously incubated, and incubated at 4 ° C. for 4 hours. After the incubation, the protein A sepharose was washed 4 times with 500 μl of buffer A, 15 μl of buffer A and 15 μl of 2 × Laemmli buffer were added, and heated at 98 ° C. for 3 minutes to form SDS. SDS-PAGE was performed in the same manner as in Example 3. However, the sample volume was 10 μl per lane. Furthermore, as in Example 3, except that Western blotting was performed using an anti-FLAG antibody as the primary antibody, the binding between Scrapper and KIF1B was detected. As a result, as shown in FIG. 5, it was shown that KIF1B co-precipitated with Scrapper only when MG132 was allowed to act, and that Scrapper and KIF1B were bound. On the other hand, coprecipitation was not observed in the absence of MG132, but it is thought that KIF1B was degraded by the proteasome after interacting with Scrapper and becoming ubiquitinated.
In addition, following the same procedure as in this example, a test substance was added to a screening system containing Scrapper and a ubiquitinated target protein, the degree of interaction between the Scrapper protein and the ubiquitinated target protein was measured, and as in MG132 It was considered that drug candidate substances could be screened by selecting a substance that promotes complex formation between them or a substance that inhibits complex formation.

Example 6: Analysis of ubiquitination of KIF5 and KIF1B
As an expression vector for KIF5, KIF5A (mouse) cDNA (SEQ ID NO: 16) added with a YFP tag was incorporated into pYFPC (BD Clontech) and used as a mammalian cell expression vector. Otherwise, in the same manner as in Example 5, Scrapper and KIF5, Scrapper and KIF1B, KIF5 or KIF1B alone (negative control) were each expressed as a protein in 293 cells, and ubiquitination was detected. Anti-GFP antibody was used as the primary antibody for Western blotting, and ubiquitination of KIF5 and KIF1B was detected. As shown in FIG. 6, the amount of KIF molecules increased in the presence of MG132 as compared to that in the absence of MG132, and in the presence of Scrapper, when Scrapper was absent in the presence or absence of MG132. Compared with the amount of KIF molecules decreased. That is, it has been clarified that degradation of the KIF molecule occurs in the ubiquitin-proteasome system and is promoted by Scrapper.
In addition, according to the same procedure as in this example, a test substance is added to a cell coexpressing Scrapper and a ubiquitinated target protein or an extract of the cell, and the degree of degradation of the ubiquitinated target protein by the proteasome is measured, It was considered that drug candidate substances can be screened by selecting a substance that reduces degradation or a substance that promotes degradation, such as MG132.

In the same manner as in Example 4, KIF1 and RIM1 were coprecipitated with Scrapper from the mouse brain using the Scrapper antibody, and then ubiquitination was detected.
Uba1 (E1), GST-UbcH5 (E2), His6-ubiquitin, APP-BP1 / Uba3, GST-UbcH12 and NEDD8 were purchased from MBL. E1, E2, His6-ubiquitin, NEDD8 system (APPBP1 / Uba3, UbcH12, NEDD8 mixture) was mixed with Scrapper antibody immunoprecipitate, and T. Kawakami et al., Embo
According to the method of J 20, 4003-12 (Aug 1, 2001)., Ubiquitination reaction was performed at 37 ° C. for 3 hours. After the reaction, SDS buffer was added to stop the reaction, and ubiquitination was detected by Western blotting. Anti-KIF1A or anti-RIM1 antibody was used as the primary antibody for Western blotting, and band shift due to ubiquitination of KIF1 and RIM1 was detected. As shown in FIG. 17, KIF1 or RIM1 co-precipitated with Scrapper serves as a substrate for ubiquitination, and a ubiquitinated KIF1 or RIM1 band is detected in a smear state at a molecular weight position larger than the original molecular weight. It was. That is, it was revealed that KIF and RIM molecules co-precipitated with Scrapper are ubiquitinated by the Scrapper complex. It is clear that ubiquitination of KIF1 and RIM1 by Scrapper is specific in those using preimmune serum (PIS).

Example 7: Localization analysis of KIF5 and KIF1 Using the Scrapper KO mouse described in Example 1, changes in the localization of KIF5 and KIF1 depending on the presence or absence of the Scrapper molecule were analyzed. Hippocampal neurons were cultured using 3-day-old Scrapper KO mice and wild-type mouse hippocampus as KO mice as controls, and 12 days later, PBS solution containing 4% paraformaldehyde was used for 15 minutes at room temperature. The cells were fixed with. The fixed cells were treated with a PBS solution containing 0.25% TritonX-100 for 5 minutes at room temperature, and then blocked by incubating with a PBS solution in which 3% BSA was dissolved for 30 minutes. As primary antibodies, KIF5 and KIF1A antibodies diluted 100-fold with a PBS solution in which 3% BSA was dissolved were used and reacted with cells at 4 ° C. overnight. The primary antibody was washed with PBS, and then reacted as a secondary antibody with Alexa488 conjugated GoatIgG anti-mouse IgG (Molecular probes) diluted 200-fold with a PBS solution in which 3% BSA was dissolved, and reacted at room temperature for 30 minutes. . The cells were further washed with PBS, embedded, and then observed with a confocal laser microscope (LSM PASCAL, manufactured by Zeiss). The results are shown in FIG. In hippocampal primary cultured neurons obtained from Scrapper KO mice, enhanced staining of KIF5 and KIF1 was observed.

Example 8: Analysis of changes in Glutamate release from synaptic vesicles by Scrapper KO
Miniature postsynaptic cu from hippocampal primary neurons obtained from Scrapper KO mice
rrent (mEPSC) was recorded (Annu Rev Physiol. 1984; 46: 455-72). The media used for mEPSC recording were 168 mM NaCl, 2.4 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM glucose, 10 mM HEPES, and 0.5 μM TTX (pH 7.3). Electrode is (4-8 MΩ) whole-cell pipette solution (120 mM K-acetate, 20 mM KCl, 0.1 mM CaCl ₂ , 5 mM MgCl ₂ , 0.2 mM EGTA, 5 mM ATP, and 10 mM HEPES (pH 7.3)) Filled with. The amplifier used was an EPC-7 amplifier (HEKA, Germany) and a Digidata 1200 acquisition board (Axon Instruments). Membrane potential is The signal was fixed at -70 mV, and the signal was filtered and recorded at 10 kHz 0.5 mV / pA for 40 sec. Cells with leakage of 250 pA or more were excluded. Membrane resistance (R _m ), series resistance (R _s ), and membrane capacitance (C _m ) were monitored. Only those with R _m > 100 MΩ and R _s <20 MΩ were analyzed (Mean R _m , R _s and C _m (P> 0.05) were compared). CNQX (50 μM, manufactured by Tocris), that is, AMPA receptor (a subtype of glutamate receptor; AMPA is an α-amino-3-hydroxyl-5-methyl-4-isoxazole propionate) antagonist. It was shown that the response is an AMPA receptor component. Recording was performed for 40 seconds. mEPSCs recorded background noise level x 3 as a footstep. Records from the same group were averaged and statistically evaluated by Student's t test (Toshihiko Igawa, “Statistics Quick Reference by Excel”, Kyoritsu Shuppan, May 2003). The results are shown in FIG. It was found that glutamate release from synaptic vesicles increased in hippocampal primary cultured neurons obtained from Scrapper KO mice. It was also found that glutamate release was increased in hippocampal slices of Scrapper KO mice.

Example 9: Analysis of changes in Glutamate release from synaptic vesicles by enhancing / blocking Scrapper activity Scrapper with a GFP tag on primary cultured cells of rat E18 hippocampus or various types of Scrapper with a GFP tag Regions (LRR region, CAXX region) were expressed and mEPSC was recorded. Expression was performed by transfection into cells on the ninth day of culture, and plasmid DNA incorporating cDNA encoding the Scrapper region shown in FIG. 9 was introduced using Lipofectamine 2000 (Invitrogen) reagent. The media used for mEPSC recording were 168 mM NaCl, 2.4 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM glucose, 10 mM HEPES, and 0.5 μM TTX (pH 7.3). Electrode is (4-8 MΩ) whole-cell pipette solution (120 mM K-acetate, 20 mM KCl, 0.1 mM CaCl ₂ , 5 mM MgCl ₂ , 0.2 mM EGTA, 5 mM ATP, and 10 mM HEPES (pH 7.3)) Filled with. The amplifier used was an EPC-7 amplifier (manufactured by HEKA, Germany) and a Digidata1200 acquisition board (manufactured by Axon Instruments). The Membrane potential was fixed at -70 mV, and the signal was filtered and recorded at 10 kHz 0.5 mV / pA for 40 sec. 250
Cells with leaks above pA were excluded. Membrane resistance (R _m ), series resistance (R _s ), and membrane capacitance (C _m ) were monitored. R _m > 100 MΩ and R _s <20
Only those with MΩ were analyzed (Mean R _m , R _s , and C _m (P> 0.05) were compared). CNQX (50 μM, manufactured by Tocris), that is, AMPA receptor antagonist, indicated that the recorded reaction was an AMPA receptor component. Recording was performed for 40 seconds. mEPSCs recorded background noise level x 3 as a footstep. Records from the same group were averaged and statistically determined by Student's t test. P <0.05 was considered significant. These measurements were made with records from cells expressing two or more independent GFPs in at least two independent cultures. EGFP-SC is a combination of EGFP and full-length Scrapper protein, EGFP-C435A is a combination of EGFP and full-length Scrapper protein in which cysteine at position 435 is substituted with alanine, EGFP-LRR is EGFP and Scrapper protein from 321 to EGFP-CAAX is a plasmid in which a partial sequence at position 380 and EGFP-CAAX are combined with a partial sequence at positions 379 to 438 of the Scrapper protein. From FIG. 9, it was found that when the wild-type Scrapper gene is overexpressed, the release of glutamic acid decreases. A similar trend was observed with EGFP-C435A, but glutamate release was increased with EGFP-LRR. This is thought to be due to LRR acting on dominant negative and inhibiting the function of endogenous Scrapper.
Wild-type Scrapper protein reduces glutamate release, suggesting that it is mediated by binding to target molecules in the LRR region.

Example 10: Identification of target molecule Neurofilament (two-dimensional electrophoresis of Tg mouse protein)
Using the Scrapper Tg mouse obtained in Example 2, the target molecule of Scrapper was identified by analyzing the quantitative change of the protein by two-dimensional electrophoresis. After the mouse was dislocated from the cervical spine, the brain was removed and weighed. Extracted brain with Polytron homogenizer together with an extraction buffer (7M urea, 2M thiourea, 2% CHAPS, 40 mM DTT, 2% IPG buffer pH3-10, Complete EDTA (-) (Roche)) 20 times the brain weight After 3000 revolutions and 10 strokes, the mixture was centrifuged at 15000 rpm for 20 minutes at 4 ° C. in a refrigerated small centrifuge. The obtained supernatant was collected, and 100 μl thereof was subjected to two-dimensional electrophoresis. The first dimension is the current focusing electrophoresis at pH 3-10, the second dimension is 12% polyacrylamide electrophoresis, the gel after the electrophoresis is stained with CBB, and the migration pattern of Tg mice and wild type mice By comparing, the changing protein was detected. Proteins were collected from the gel for the spots with changes, MS analysis or peptide sequencing was performed to determine the protein sequence, and the proteins with changes were identified. As a result, it was found that Neurofilament-M and -L were decreased in Tg mice (FIG. 10).

Example 11: Identification of the target molecule Neurofilament (Western blot of Tg and KO mouse proteins)
Using the Tg mouse and KO mouse obtained in Example 1 and Example 2, the target molecule was identified by analyzing the quantitative change of the protein by Western blot. After the mouse was dislocated from the cervical spine, the brain was removed and weighed. Extracted brain with Polytron homogenizer together with an extraction buffer (7M urea, 2M thiourea, 2% CHAPS, 40 mM DTT, 2% IPG buffer pH3-10, Complete EDTA (-) (Roche)) 20 times the brain weight After 3000 revolutions and 10 strokes, the mixture was centrifuged at 15000 rpm for 20 minutes at 4 ° C. in a refrigerated small centrifuge. The obtained supernatant was collected, and protein quantification was performed by the Bradford method (Bio-Rad Protein Assay Dye Regent Concentrate, manufactured by Bio-Rad). Based on the quantified protein concentration, an SDS sample was prepared so as to be 0.5 μg / μl or 0.05 μg / μl. SDS was performed by the method of Laemmli et al. After applying 10 μl of each sample to each lane using 8% polyacrylamide gel by the method of Laemmli et al., SDS-PAGE was performed, and the protein in the gel was further transferred to a PVDF membrane (Millipore). Transcription was performed according to Trnovsky et al. Biotechniques 13 (5), 800-804 (1992). Next, the PVDF membrane was subjected to Western blotting. After blocking for 1 hour in TBST (50 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween20) in which 5% skim milk (Wako) was dissolved, the anti-NF-H antibody (Sigma) was used as the primary antibody. ), Anti-NF-M antibody (manufactured by Sigma), and anti-NF-L antibody (manufactured by Sigma), each at a concentration of 1 μg / ml and incubated overnight at 4 ° C. for reaction. After the reaction, the membrane was washed with TBST, and reacted for 30 minutes at room temperature in a solution diluted with a peroxidase-conjugated anti-mouse or anti-goat antibody (Jackson) 5000 times. After the membrane was again washed with TBST, a peroxidase luminescent substrate (Western blotting detection regents: manufactured by Amersham) was added to perform a chemiluminescent reaction, and the film (Hyperfilm, manufactured by Amersham) was exposed to light and detected. By this operation, the amounts of NF-M, NF-H, and NF-L proteins in Scrapper Tg mice and KO mice were detected, and the amounts were compared with those of normal mice. The results are shown in FIG. The result of Western blotting is shown in the upper part of FIG. 11 and the band density is quantified in the lower part of FIG. It was detected that Neurofilament molecules were increased in KO mice than in normal mice and decreased in Tg mice. In Tg mice, the overexpression of Scrapper is thought to decrease because more Neurofilament molecules are destroyed. That is, it was shown that Scrapper has a function of degrading Neurofilament molecules, and Neurofilament is a target molecule of Scrapper.

Example 12: Analysis of interaction between Scrapper and Neurofilament (immunoprecipitation from mouse brain)
In order to examine the interaction between Scrapper and Neurofilament, immunoprecipitation was performed from the brain of a mouse (C57BL / 6J or ICR) using Scrapper antibody. After cervical dislocation of 3 mice, the brain was removed, homogenized with 10 ml buffferA (50 mM Tris pH 8.0, 100 mM NaCl, 1% TritonX-100, 10 μg / ml leupeptin, 10 μg / ml PMSF), at 4 ° C., Centrifugation was performed at 100,000 × g for 30 minutes to obtain a mouse brain extract. Divide the solution into two equal parts, each with anti-Scrapper antibody 10
10 μl of protein A sepharose (Amersham) preincubated with μl, 10 μl of protein A sepharose (Amersham) preincubated with anti-Scrapper antibody preimmune serum as a negative control, and incubated overnight at 4 ° C. . After incubation, protein A sepharose was washed 4 times with 1 ml of buffer A, 50 μl of buffer A and 50 μl of 2 × Laemmli buffer were added, and the mixture was heated at 98 ° C. for 3 minutes for SDS conversion. SDS-PAGE was performed in the same manner as in Example 4. However, the sample volume was 10 μl per lane. Furthermore, according to Example 3, Western blotting was performed using anti-Neurofilament-H antibody, anti-Neurofilament-M antibody and anti-Neurofilament-L antibody, and the binding of Scrapper, Neurofilament-H and Neurofilament-M in the mouse brain was detected. . As a result, as shown in FIG. 12, Neurofilament-H and Neurofilament-M co-precipitated with Scrapper, and it was shown that Neurofilament-H and Neurofilament-M bind to Scrapper. Although binding to Neurofilament-L could not be confirmed by Western blotting, this was not directly detected by Neurofilament-L, or the binding was weak, so even if it was bound, it was less than the detection sensitivity in this experimental system. It is thought that it was because of.

Example 13: Identification of the target molecule Myosin (two-dimensional electrophoresis and Western blotting of KO and Tg mouse proteins)
Using the Scrapper KO mouse and Tg mouse obtained in Example 1 and Example 2, the target molecule was identified by analyzing the quantitative change of the protein by two-dimensional electrophoresis and Western blot.
After the mouse was dislocated from the cervical spine, the brain was removed and weighed. Extracted brain with Polytron homogenizer together with an extraction buffer (7M urea, 2M thiourea, 2% CHAPS, 40 mM DTT, 2% IPG buffer pH3-10, Complete EDTA (-) (Roche)) 20 times the brain weight After 3,000 revolutions and 10 strokes, the mixture was centrifuged at 15,000 rpm for 20 minutes at 4 ° C. in a cooling small centrifuge. The obtained supernatant was collected, and protein quantification was performed by the Bradford method (Bio-Rad Protein Assay Dye Regent Concentrate, manufactured by Bio-Rad). Based on the quantified protein concentration, an SDS sample was prepared so as to be 0.5 μg / μl or 0.05 μg / μl. SDS was performed by the method of Laemmli et al. After applying 10 μl of each sample to each lane using 8% polyacrylamide gel by the method of Laemmli et al., SDS-PAGE was performed, and the protein in the gel was further transferred to a PVDF membrane (Millipore). Transcription was performed according to Trnovsky et al. Biotechniques 13 (5), 800-804 (1992). Next, the PVDF membrane was subjected to Western blotting. After blocking with TBST (50 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween 20) dissolved in 5% skim milk (Wako) for 1 hour, anti-Myosin IIB antibody (Sigma) was used as the primary antibody. In the presence of each of them at a concentration of 1 μg / ml overnight at 4 ° C. for reaction. After the reaction, the membrane was washed with TBST, and reacted for 30 minutes at room temperature in a solution diluted with a peroxidase-conjugated anti-mouse or anti-goat antibody (Jackson) 5000 times. After the membrane was again washed with TBST, a peroxidase luminescent substrate (Western blotting detection regents: manufactured by Amersham) was added to perform a chemiluminescent reaction, and the film (Hyperfilm, manufactured by Amersham) was exposed to light and detected. By this operation, the amount of MyosinIIB protein in Scrapper mutant mice was detected, and the amount was compared with that of normal mice. The results are shown in FIG. The result of Western blotting is shown in the upper part of FIG. 13, and the band density is shown in the lower part of FIG. It was detected that MyosinIIB molecules increased in KO mice than in normal mice and decreased in Tg mice. In KO mice, Scrapper deficiency increases Myosin molecules compared to normal mice, and in Tg mice, Scrapper overexpression is thought to decrease due to more Myosin molecules being destroyed. That is, it was shown that Scrapper has a function of decomposing Myosin molecule, and MyosinIIB is a target molecule of Scrapper.

Example 14: Analysis of interaction between Scrapper and Myosin (immunoprecipitation from mouse brain)
In order to examine the interaction between Scrapper and Myosin, immunoprecipitation was performed from the brain of a mouse (C57BL / 6J or ICR) using the Scrapper antibody. After cervical dislocation of 3 mice, the brain was removed and homogenized with 10 ml of buffferA (50 mM Tris pH 8.0, 100 mM NaCl, 1% TritonX-100, 10 μg / ml leupeptin, 10 μg / ml PMSF) at 4 ° C., Centrifugation was performed at 100,000 × g for 30 minutes to obtain a mouse brain extract. The solution was divided into two equal parts, each 10 μl of proteinA sepharose (manufactured by Amersham) pre-incubated with 10 μl of anti-Scrapper antibody, and proteinA sepharose preincubated with pre-immune serum of anti-Scrapper antibody as a negative control. (Amersham) was mixed with 10 μl and incubated at 4 ° C. overnight. After incubation, protein A sepharose was washed 4 times with 1 ml of buffer A, 50 μl of buffer A and 50 μl of 2 × Laemmli buffer were added, and the mixture was heated at 98 ° C. for 3 minutes for SDS conversion. SDS-PAGE was performed in the same manner as in Example 3. However, the sample volume was 10 μl per lane. Further, Western blotting was performed in the same manner as in Example 3 to detect the binding of Scrapper, MyosinIIB, and MyosinVa in the mouse brain. As shown in FIG. 14, the results showed that MyosinIIB and MyosinVa co-precipitated with Scrapper, and MyosinIIB and MyosinVa bound to Scrapper. In contrast, MyosinIIA is not co-precipitated, and it is clear that the binding of MyosinIIB, MyosinVa and Scrapper is specific.

In addition, 293T cells expressing FLAG-Scrapper fusion protein were used to examine the ubiquitination of MyosinIIB by Scrapper and the accompanying degradation by the proteasome (FIG. 15). As a result of immunoprecipitation using an anti-FLAG antibody and Western blotting with an anti-Myosin IIB antibody, coprecipitation of Scrapper and Myosin IIB was observed when the proteasome inhibitor MG132 was added. On the other hand, coprecipitation was not observed in the absence of the proteasome inhibitor MG132, but it is thought that MyosinIIB was ubiquitinated by interacting with Scrapper and then degraded by the proteasome.
In addition, following the same procedure as in this example, a test substance was added to a screening system containing Scrapper and a ubiquitinated target protein, the degree of interaction between the Scrapper protein and the ubiquitinated target protein was measured, and as in MG132 It was considered that drug candidate substances could be screened by selecting a substance that promotes complex formation between them or a substance that inhibits complex formation.

Example 15: Analysis of Scrapper Transcription Induction Induction of Scrapper mRNA Expression in Primary Hippocampal Culture Cells by Forskolin Stimulation Forskolin stimulation is a method described in Sala et al. J Neurosci. 2000 May 15; 20 (10): 3529-36. Went according to. Specifically, first, mouse hippocampal primary cultured cells were treated with 50 μM forskolin. To suppress endogenous synaptic activity, 1 μM tetrodotoxin was added 12 hours prior to forskolin stimulation. Forskolin was added to the medium and maintained in a 5% CO ₂ incubator. After 0, 1, 3, 6, and 12 hours, Total RNA was isolated using Sepasol reagent (manufactured by Nacalaitrsque). CDNA was synthesized from 1 μg of total RNA using ReverTra Ace (TOYOBO) and used as a template for PCR. Scrapper mRNA was quantified using 2100Bioanalyzer (Agilent).
The results are shown in FIG. It was found that Scrapper expression was induced by forskolin stimulation.

Example 16: Analysis of change in release amount of Glutamate from synaptic vesicles by MG132 (proteasome inhibitor)
Miniature postsynaptic current (mEPSC) was recorded after adding 50 μM MG132 to hippocampal primary cultured neurons obtained from Wistar rats and incubating for 1 hour (Annu Rev Physiol. 1984; 46: 455-72). Measurement was also performed when MG132 was not added as a control. The media used for mEPSC recording were 168 mM NaCl, 2.4 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 10
mM glucose, 10 mM HEPES, and 0.5 μM TTX (pH 7.3). The electrode is (4-8 MΩ) whole-cell pipette solution (120 mM K-acetate, 20 mM KCl, 0.1 mM CaCl ₂ , 5 mM MgCl ₂ , 0.2
It was filled with mM EGTA, 5 mM ATP, and 10 mM HEPES (pH 7.3)). The amplifier used was an EPC-7 amplifier (HEKA, Germany) and a Digidata 1200 acquisition board (Axon Instruments). Membrane potential is The signal was fixed at -60 mV, and the signal was filtered and recorded at 10 kHz 0.5 mV / pA for 40 sec. Cells with leakage of 250 pA or more were excluded. Membrane resistance (R _m ), series resistance (R _s ), and membrane capacitance (C _m ) were monitored. Only those with R _m > 100 MΩ and R _s <20 MΩ were analyzed (Mean R _m , R _s and C _m (P> 0.05) were compared). CNQX (50 μM, manufactured by Tocris), that is, AMPA receptor (a subtype of glutamate receptor; AMPA is an α-amino-3-hydroxyl-5-methyl-4-isoxazole propionate) antagonist. It was shown that the response is an AMPA receptor component. Recording was performed for 40 seconds. mEPSCs recorded background noise level x 3 as a footstep. Records from the same group were averaged and statistically evaluated by Student's t test (Toshihiko Igawa, “Statistics Quick Reference by Excel”, Kyoritsu Shuppan, May 2003).
The results are shown in FIG. It was found that glutamate release from synaptic vesicles increased by the addition of MG132 in primary cultured hippocampal neurons obtained from rats. In the absence of CaCl ₂ , glutamate release was not increased even when MG132 was added.
Similarly, in the hippocampal primary cultured neurons obtained from mice, the result that MG132 promotes the release of glutamate was obtained.
Based on these results, test substances are added to non-human mammals that express the Scrapper gene or biological samples obtained from these animals, and the release of neurotransmitters is measured to accelerate the release of neurotransmitters like MG132. It can be seen that screening of drug candidate substances is possible by selecting substances that inhibit the release of substances to be transmitted and neurotransmitters.

The figure which shows the procedure of preparation of a Scrapper gene knockout mouse (partial photograph). The upper part shows the targeting vector, the lower left part shows RT-PCR, and the lower right part shows the result of Western blotting. The figure which shows the procedure of preparation of a Scrapper gene transgenic mouse (partial photograph). The upper shows transgene, the lower left shows RT-PCR, and the lower right shows Western blotting results. The figure which shows the expression of KIF in a Scrapper gene knockout mouse and a transgenic mouse (partial photograph). The upper graph shows the results of Western blotting, and the lower graph shows the expression level. Diagram (photo) showing the interaction between Scrapper and KIF or RIM1. Input represents an antibody-untreated sample, PISIP represents an immunoprecipitate using a pre-immune serum, and SC IP represents an immunoprecipitate using a Scrapper antibody. Western blotting was performed with each KIF antibody, RIM1 antibody or Dynein antibody. Figure 2 showing the interaction between Scrapper and KIF (photo). Immunoprecipitation was performed with GFP antibody, and Western blotting was performed with FLAG antibody. Diagram (photo) showing the disassembly of KIF by Scrapper. Immunoprecipitation with GFP antibody and Western blotting with GFP antibody were performed. Diagram showing the localization of KIF in Scrapper knockout mice (-/-) hippocampal primary cells. (+ / +) Shows the results of primary cultured cells of wild type mouse hippocampus. The figure which shows the release | release of glutamic acid from the synaptic vesicle in the knockout mouse (-/-) hippocampus primary culture cell of Scrapper. (+ / +) Shows the results of primary cultured cells of wild type mouse hippocampus. The figure which shows discharge | release of glutamic acid from a synaptic vesicle when each Scrapper gene is expressed in the hippocampal primary culture cell (part photograph). A shows the structure of each Scrapper gene construct. B shows the intracellular localization of each Scrapper gene product. MAP2 shows the result of staining with an anti-MAP2 antibody, Merge shows the result of overlaying the fluorescence image of GFP and the result of staining of MAP2, and the arrows show axons. Since MAP2 is a marker of dendrite, it indicates that wild-type Scrapper protein is localized in axons. C shows a record (trace) of mEPSC when each Scrapper gene product is expressed in nerve cells. D is a graph of mEPSC frequency and amplitude. The figure (photograph) which shows the comparison by the two-dimensional electrophoresis of the brain protein in a wild type mouse | mouth and a Scrapper gene transgenic mouse. The figure which shows the expression of each neurofilament in a Scrapper gene transgenic mouse (partial photograph). The upper graph shows the results of Western blotting, and the lower graph shows the expression level. A diagram (photo) showing the interaction between Scrapper and each neurofilament. Input represents an antibody-untreated sample, PISIP represents an immunoprecipitate using a pre-immune serum, and SC IP represents an immunoprecipitate using a Scrapper antibody. Western blotting was performed with each neurofilament antibody. The figure which shows the expression of myosin in a Scrapper gene knockout mouse and a transgenic mouse (partial photograph). The upper graph shows the results of Western blotting, and the lower graph shows the expression level. Diagram showing the interaction between Scrapper and each myosin (photo). Input represents an antibody-untreated sample, PISIP represents an immunoprecipitate using a pre-immune serum, and SC IP represents an immunoprecipitate using a Scrapper antibody. Western blotting was performed with each myosin antibody. Diagram (photo) showing ubiquitination of myosin IIB by Scrapper. The figure which shows the expression induction of Scrapper by forskolin stimulation (partial photograph). The left figure shows the results of electrophoresis of RT-PCR products, and the right figure is a graph showing the expression level. SC is the Scrapper gene, G6PDH is the glucose-6-phosphate dehydrogenase gene, P-CREB is the phosphorylated cAMP response element (CRE) -binding protein (CREB), and CREB is the cAMP response element (CRE) -binding Protein is shown. It is known that phosphorylation of CREB is caused by forskolin, and phosphorylated CREB increases after forskolin stimulation, indicating that forskolin stimulation was performed appropriately. At this time, it is clear from the RT-PCR results that the transcription of the Scrapper gene is induced and the mRNA is increased. The figure which shows ubiquitination of KIF1 and RIM1 by Scrapper. Input represents an antibody-untreated sample, PIS represents an immunoprecipitate using preimmune serum, and SCR represents an immunoprecipitate using a Scrapper antibody. IP + Ubi indicates the case where ubiquitinase complex of E1, E2, His6-ubiquitin, NEDD8 system is added. The figure which shows the release analysis of Glutamate from the synaptic vesicle by MG132 (proteasome inhibitor).

Claims (14)

A ubiquitination target protein selected from a cytoskeletal protein and a synapse-related protein, and a test substance added to a screening system containing a Scrapper protein, and measuring the degree of ubiquitination of the ubiquitination target protein Candidate substance screening method. The screening method according to claim 1, wherein the screening system is a cell that co-expresses the ubiquitination target protein and the Scrapper protein or an extract of the cell. The screening method according to claim 1, wherein the screening system is an in vitro system containing the ubiquitinated target protein and the Scrapper protein. The screening method according to claim 1, wherein the screening system is a non-human mammal expressing a Scrapper protein or a biological sample obtained from the animal. A test substance is added to a ubiquitinated target protein selected from cytoskeletal proteins and synapse-related proteins, and cells that co-express Scrapper protein, or the extract of the cells, and the degree of degradation of the ubiquitinated target protein by the proteasome is measured. A method for screening a drug candidate substance. A test substance is added to a non-human mammal expressing Scrapper protein or a biological sample obtained from the animal, and the proteasome degrades a ubiquitinated target protein selected from cytoskeletal proteins and synapse-related proteins in the animal or biological sample A method for screening a drug candidate substance, which comprises measuring the degree of the drug. A test substance is added to a screening system containing a ubiquitinated target protein selected from cytoskeletal proteins and synapse-related proteins, and a scrapper protein, and the degree of interaction between the scrapper protein and the ubiquitinated target protein is measured. A method for screening a drug candidate substance. The screening method according to any one of claims 1 to 7, wherein the ubiquitinated target protein is KIF. The screening method according to any one of claims 1 to 7, wherein the ubiquitinated target protein is a neurofilament. The screening method according to any one of claims 1 to 7, wherein the ubiquitinated target protein is myosin. The screening method according to any one of claims 1 to 7, wherein the ubiquitinated target protein is RIM. The screening method according to any one of claims 1 to 11, wherein the drug candidate substance is a candidate substance for a therapeutic or preventive drug for neurodegenerative disease or schizophrenia. A screening method for a drug candidate substance, comprising adding a test substance to a nerve cell in which the Scrapper gene is expressed, and measuring the expression level of the Scrapper gene or the release of a neurotransmitter. Screening for drug candidates, comprising adding a test substance to a non-human mammal expressing the Scrapper gene or a biological sample obtained from the animal, and measuring the expression level of the Scrapper gene or the release of a neurotransmitter Method.

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